Piperidinyl compound as a modulator of chemokine receptor activity

ABSTRACT

The present application describes the compound of formula (I): 
     
       
         
         
             
             
         
       
     
     or stereoisomers or pharmaceutically acceptable salts thereof. In addition, methods of treating and preventing inflammatory diseases such as asthma and allergic diseases, as well as autoimmune pathologies such as rheumatoid arthritis and arthrosclerosis using the compound of the invention are disclosed.

This application claims the benefit of U.S. Provisional Application Ser.No. 61/355,225 filed on Jun. 16, 2010, which is hereby incorporated byreference in its entirety.

FIELD OF THE INVENTION

This invention relates generally to a piperidinyl compound which is amodulator of chemokine receptor activity, pharmaceutical compositionscontaining said compound, and methods of using the compound as an agentfor treatment and prevention of inflammatory diseases, allergic andautoimmune diseases, and in particular, rheumatoid arthritis andtransplant rejection.

BACKGROUND OF THE INVENTION

Chemokines are chemotactic cytokines, of molecular weight 6-15 kDa, thatare released by a wide variety of cells to attract and activate, amongother cell types, monocytes, macrophages, T and B lymphocytes,eosinophils, basophils and neutrophils. There are two major classes ofchemokines, CXC and CC, depending on whether the first two cysteines inthe amino acid sequence are separated by a single amino acid (CXC) orare adjacent (CC). The CXC chemokines, such as interleukin-8 (IL-8),neutrophil-activating protein-2 (NAP-2) and melanoma growth stimulatoryactivity protein (MGSA) are chemotactic primarily for neutrophils and Tlymphocytes, whereas the CC chemokines, such as RANTES, MIP-1α, MIP-1β,the monocyte chemotactic proteins (MCP-1, MCP-2, MCP-3, MCP-4, andMCP-5) and the eotaxins (−1 and −2) are chemotactic for, among othercell types, macrophages, T lymphocytes, eosinophils, dendritic cells,and basophils.

The chemokines bind to specific cell-surface receptors belonging to thefamily of G-protein-coupled seven-transmembrane-domain proteins whichare termed “chemokine receptors.” On binding their cognate ligands,chemokine receptors transduce an intracellular signal though theassociated trimeric G proteins, resulting in, among other responses, arapid increase in intracellular calcium concentration, changes in cellshape, increased expression of cellular adhesion molecules,degranulation, and promotion of cell migration. There are at least tenhuman chemokine receptors that bind or respond to CC chemokines with thefollowing characteristic patterns: CCR-1 (or “CKR-1” or “CC-CKR-1”)[MIP-1α, MCP-3, MCP-4, RANTES] CCR-2A and CCR-2B (or “CKR-2A”/“CKR-2B”or “CC-CKR-2A”/“CC-CKR-2B”) [MCP-1, MCP-2, MCP-3, MCP-4, MCP-5]; CCR-3(or “CKR-3” or “CC-CKR-3”) [eotaxin-1, eotaxin-2, RANTES, MCP-3, MCP-4];CCR-4 (or “CKR-4” or “CC-CKR-4”) [TARC, MDC]; CCR-5 (or “CKR-5” OR“CC-CKR-5”) [MIP-1α, RANTES, MIP-1β]; CCR-6 (or “CKR-6” or “CC-CKR-6”)[LARC]; CCR-7 (or “CKR-7” or “CC-CKR-7”) [ELC]; CCR-8 (or “CKR-8” or“CC-CKR-8”) [1-309]; CCR-10 (or “CKR-10” or “CC-CKR-10”) [MCP-1, MCP-3];and CCR-11 [MCP-1, MCP-2, and MCP-4].

In addition to the mammalian chemokine receptors, mammaliancytomegaloviruses, herpesviruses and poxviruses have been shown toexpress, in infected cells, proteins with the binding properties ofchemokine receptors. Human CC chemokines, such as RANTES and MCP-3, cancause rapid mobilization of calcium via these virally encoded receptors.Receptor expression may be permissive for infection by allowing for thesubversion of normal immune system surveillance and response toinfection. Additionally, human chemokine receptors, such as CXCR4, CCR2,CCR3, CCR5 and CCR8, can act as co-receptors for the infection ofmammalian cells by microbes as with, for example, the humanimmunodeficiency viruses (HIV).

The chemokines and their cognate receptors have been implicated as beingimportant mediators of inflammatory, infectious, and immunoregulatorydisorders and diseases, including asthma and allergic diseases, as wellas autoimmune pathologies such as rheumatoid arthritis andarthrosclerosis (reviewed in: Carter, P. H., Current Opinion in ChemicalBiology 2002, 6, 510; Trivedi et al., Ann. Reports Med. Chem. 2000, 35,191; Saunders et al., Drug Disc. Today 1999, 4, 80; Premack et al.,Nature Medicine 1996, 2, 1174). For example, the chemokine macrophageinflammatory protein-1 (MIP-1a) and its receptor CC Chemokine Receptor 1(CCR-1) play a pivotal role in attracting leukocytes to sites ofinflammation and in subsequently activating these cells. When thechemokine MIP-1α binds to CCR-1, it induces a rapid increase inintracellular calcium concentration, increased expression of cellularadhesion molecules, cellular degranulation, and the promotion ofleukocyte migration.

In addition, demonstration of the chemotactic properties of MIP-1α inhumans has been provided experimentally. Human subjects, when injectedintradermally with MIP-1α, experienced a rapid and significant influx ofleukocytes to the site of injection (Brummet, M. E., J. Immun. 2000,164, 3392-3401).

It is known that MIP-1α is elevated in the synovial fluid and blood ofpatients with rheumatoid arthritis. Moreover, several studies havedemonstrated the potential therapeutic value of antagonism of theMIP-1a/CCR1 interaction in treating rheumatoid arthritis.

It should also be noted that CCR-1 is also the receptor for thechemokines RANTES, MCP-3, HCC-1, Lkn-1/HCC-2, HCC-4, and MPIF-1 (Carter,P. H., Curr. Opin Chem. Bio. 2002, 6, 510-525). Since it is presumedthat the new compound of formula (I) described herein antagonizes MIP-1αby binding to the CCR-1 receptor, it may be that this compound is alsoan effective antagonist of the actions of the aforementioned ligand thatare mediated by CCR-1. Accordingly, when reference is made herein to“antagonism of MIP-1α,” it is to be assumed that this is equivalent to“antagonism of chemokine stimulation of CCR-1.”

Recently, a number of groups have described the development of smallmolecule antagonists of MIP-1α (reviewed in: Carson, K. G. et al., Ann.Reports Med. Chem. 2004, 39, 149-158).

SUMMARY OF THE INVENTION

Accordingly, the present invention provides a piperidinyl compound whichis an antagonist or partial agonist/antagonist of MIP-1α or CCR-1receptor activity, or a pharmaceutically acceptable salt thereof.

The present invention provides pharmaceutical compositions comprising apharmaceutically acceptable carrier and a therapeutically effectiveamount of the compound of the present invention or a pharmaceuticallyacceptable salt form thereof.

The present invention provides a method for treating rheumatoidarthritis and transplant rejection, comprising administering to a hostin need of such treatment a therapeutically effective amount of thecompound of the present invention or a pharmaceutically acceptable saltform thereof.

The present invention provides a method for treating inflammatorydiseases, comprising administering to a host in need of such treatment atherapeutically effective amount of the compounds of the presentinvention or a pharmaceutically acceptable salt form thereof.

The present invention provides a compound for use in therapy.

The present invention provides the use of a compound for the manufactureof a medicament for the treatment of inflammatory diseases.

DETAILED DESCRIPTION OF THE INVENTION

In one embodiment, the present invention provides a compound of formula(I):

or a stereoisomer or pharmaceutically acceptable salt thereof.

The present invention provides a compound that has an unexpectedlyadvantageous profile as compared to known inhibitors of CCR-1 activity,for example, the compounds described in U.S. Pat. No. 7,601,844, andassigned to applicant. More preferably, the compound exhibits a superiorpharmacokinetic profile and other properties that make it an attractivecandidate for clinical development. Accordingly, it is these unexpectedproperties alone and/or in combination that make the compound of formula(I) desirable for use as a pharmaceutical agent.

In another embodiment, the present invention is directed to apharmaceutical composition, comprising a pharmaceutically acceptablecarrier and a therapeutically effective amount of a compound of formula(I).

In another embodiment, the present invention is directed to a method formodulation of chemokine or chemokine receptor activity comprisingadministering to a patient in need thereof a therapeutically effectiveamount of a compound of formula (I).

In another embodiment, the present invention is directed to a method formodulation of CCR-1 receptor activity comprising administering to apatient in need thereof a therapeutically effective amount of a compoundof formula (I).

In another embodiment, the present invention is directed to a method formodulation of MIP-1α, MCP-3, MCP-4, RANTES activity, preferablymodulation of MIP-1α activity, that is mediated by the CCR-1 receptorcomprising administering to a patient in need thereof a therapeuticallyeffective amount of a compound of formula (I).

In another embodiment, the present invention is directed to a method fortreating disorders, comprising administering to a patient in needthereof a therapeutically effective amount of a compound of formula (I),wherein said disorder is selected from osteoarthritis, aneurysm, fever,cardiovascular effects, Crohn's disease, congestive heart failure,autoimmune diseases, HIV-infection, HIV-associated dementia, psoriasis,idiopathic pulmonary fibrosis, transplant arteriosclerosis, physically-or chemically-induced brain trauma, neuropathic pain, inflammatory boweldisease, alveolitis, ulcerative colitis, systemic lupus erythematosus,nephrotoxic serum nephritis, glomerulonephritis, asthma, multiplesclerosis, arthrosclerosis, rheumatoid arthritis, restenosis, organtransplantation, psoriatic arthritis, multiple myeloma, allergies, suchas skin and mast cell degranulation in eye conjunctiva, hepatocellularcarcinoma, colorectal cancer, osteoporosis, renal fibrosis, and othercancers. Preferably, the invention is directed to a method of treatingCrohn's disease, psoriasis, inflammatory bowel disease, systemic lupuserythematosus, multiple sclerosis, rheumatoid arthritis, multiplemyeloma, allergies, such as skin and mast cell degranulation in eyeconjunctiva, hepatocellular carcinoma, osteoporosis or renal fibrosis.

In another embodiment, the present invention is directed to a method fortreating inflammatory diseases, comprising administering to a patient inneed thereof a therapeutically effective amount of a compound of formula(I).

In another embodiment, the present invention is directed to a method fortreating inflammatory diseases, for example, inflammatory diseases whichare at least partially mediated by CCR-1, comprising administering to apatient in need thereof a therapeutically effective amount of a compoundof formula (I).

In another embodiment, the present invention is directed to a method formodulation of CCR1 activity comprising administering to a patient inneed thereof a therapeutically effective amount of a compound of formula(I).

In another embodiment, the present invention is directed the use of acompound of formula (I) in the preparation of a medicament for thetreatment of a disorder, said disorder is selected from osteoarthritis,aneurysm, fever, cardiovascular effects, Crohn's disease, congestiveheart failure, autoimmune diseases, HIV-infection, HIV-associateddementia, psoriasis, idiopathic pulmonary fibrosis, transplantarteriosclerosis, physically- or chemically-induced brain trauma,neuropathic pain, inflammatory bowel disease, alveolitis, ulcerativecolitis, systemic lupus erythematosus, nephrotoxic serum nephritis,glomerulonephritis, asthma, multiple sclerosis, arthrosclerosis,rheumatoid arthritis, restenosis, organ transplantation, psoriaticarthritis, multiple myeloma, allergies, such as skin and mast celldegranulation in eye conjunctiva, hepatocellular carcinoma, colorectalcancer, osteoporosis, renal fibrosis and other cancers. Preferably, thepresent invention is directed the use of a compound of formula (I) inthe preparation of a medicament for the treatment of Crohn's disease,psoriasis, inflammatory bowel disease, systemic lupus erythematosus,multiple sclerosis, rheumatoid arthritis, multiple myeloma, allergies,such as skin and mast cell degranulation in eye conjunctiva,hepatocellular carcinoma, osteoporosis or renal fibrosis.

In another embodiment, the present invention is directed to a compoundof formula (I) for use in therapy.

In another embodiment, the present invention is directed to apharmaceutical composition comprising a compound of formula (I) and oneor more active ingredients.

In another embodiment, the present invention is directed to a method formodulation of chemokine or chemokine receptor activity comprisingadministering to a patient in need thereof a therapeutically effectiveamount of a pharmaceutical composition comprised of a compound offormula (I) and one or more active ingredients.

In another embodiment, the present invention is directed to a method formodulation of CCR-1 receptor activity comprising administering to apatient in need thereof a therapeutically effective amount of apharmaceutical composition comprised of a compound of formula (I) andone or more active ingredients.

In yet another embodiment, the present invention is directed to a methodfor modulation of MIP-1α, MCP-3, MCP-4 or RANTES activity, preferablymodulation of MIP-1α activity, that is mediated by the CCR-1 receptor,comprising administering to a patient in need thereof a therapeuticallyeffective amount of a pharmaceutical composition comprised of a compoundof formula (I) and one or more active ingredients.

In another embodiment, the present invention is directed to a method fortreating a disorder, comprising administering to a patient in needthereof a therapeutically effective amount of a pharmaceuticalcomposition comprised of a compound of formula (I) and one or moreactive ingredients, wherein said disorder is selected fromosteoarthritis, aneurysm, fever, cardiovascular effects, Crohn'sdisease, congestive heart failure, autoimmune diseases, HIV-infection,HIV-associated dementia, psoriasis, idiopathic pulmonary fibrosis,transplant arteriosclerosis, physically- or chemically-induced braintrauma, neuropathic pain, inflammatory bowel disease, alveolitis,ulcerative colitis, systemic lupus erythematosus, nephrotoxic serumnephritis, glomerulonephritis, asthma, multiple sclerosis,arthrosclerosis, rheumatoid arthritis, restenosis, organtransplantation, psoriatic arthritis, multiple myeloma, allergies, suchas skin and mast cell degranulation in eye conjunctiva, hepatocellularcarcinoma, colorectal cancer, osteoporosis, renal fibrosis and othercancers, preferably, Crohn's disease, psoriasis, inflammatory boweldisease, systemic lupus erythematosus, multiple sclerosis, rheumatoidarthritis, multiple myeloma, allergies, for example, skin and mast celldegranulation in eye conjunctiva, hepatocellular carcinoma, osteoporosisand renal fibrosis.

In yet another embodiment, the present invention, is directed to amethod for treating inflammatory diseases, preferably, inflammatorydiseases which are at least partially mediated by CCR-1, comprisingadministering to a patient in need thereof a therapeutically effectiveamount of a pharmaceutical composition comprised of a compound offormula (I) and one or more active ingredients.

In another embodiment, the present invention is directed to a method formodulation of CCR-1 activity comprising administering to a patient inneed thereof a therapeutically effective amount of a pharmaceuticalcomposition comprised of a compound of formula (I) and one or moreactive ingredients.

In another embodiment, the present invention is directed to the use of apharmaceutical composition comprised of a compound of formula (I) andone or more active ingredients in the preparation of a medicament forthe treatment of a disorder, said disorder is selected fromosteoarthritis, aneurysm, fever, cardiovascular effects, Crohn'sdisease, congestive heart failure, autoimmune diseases, HIV-infection,HIV-associated dementia, psoriasis, idiopathic pulmonary fibrosis,transplant arteriosclerosis, physically- or chemically-induced braintrauma, neuropathic pain, inflammatory bowel disease, alveolitis,ulcerative colitis, systemic lupus erythematosus, nephrotoxic serumnephritis, glomerulonephritis, asthma, multiple sclerosis,arthrosclerosis, rheumatoid arthritis, restenosis, organtransplantation, psoriatic arthritis, multiple myeloma, allergies, suchas skin and mast cell degranulation in eye conjunctiva, hepatocellularcarcinoma, colorectal cancer, osteoporosis, renal fibrosis and othercancers, preferably, Crohn's disease, psoriasis, inflammatory boweldisease, systemic lupus erythematosus, multiple sclerosis, rheumatoidarthritis, multiple myeloma, allergies, for example, skin and mast celldegranulation in eye conjunctiva, hepatocellular carcinoma, osteoporosisand renal fibrosis.

In still yet another embodiment, the present invention is directed tothe use of a pharmaceutical composition comprised of a compound offormula (I) and one or more active ingredients in therapy.

The invention may be embodied in other specific forms without departingfrom the spirit or essential attributes thereof. This invention alsoencompasses all combinations of alternative aspects of the inventionnoted herein. It is understood that any and all embodiments of thepresent invention may be taken in conjunction with any other embodimentto describe additional embodiments of the present invention.Furthermore, any elements of an embodiment may be combined with any andall other elements from any of the embodiments to describe additionalembodiments.

DEFINITIONS

The compound herein described may have asymmetric centers. The compoundof the present invention containing an asymmetrically substituted atommay be isolated in optically active or racemic forms. It is well knownin the art how to prepare optically active forms, such as by resolutionof racemic forms or by synthesis from optically active startingmaterials. Many geometric isomers of olefins, C═N double bonds, and thelike can also be present in the compounds described herein, and all suchstable isomers are contemplated in the present invention. Cis and transgeometric isomers of the compounds of the present invention aredescribed and may be isolated as a mixture of isomers or as separatedisomeric forms. All chiral, diastereomeric, racemic forms and allgeometric isomeric forms of a structure are intended, unless thespecific stereochemistry or isomeric form is specifically indicated.

One enantiomer of a compound of Formula I may display superior activitycompared with the other. Thus, all of the stereochemistries areconsidered to be a part of the present invention. When required,separation of the racemic material can be achieved by HPLC using achiral column or by a resolution using a resolving agent as known to oneof ordinary skill in the art.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose compounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

As used herein, “pharmaceutically acceptable salts” refer to derivativesof the disclosed compound wherein the parent compound is modified bymaking acid or base salts thereof. Examples of pharmaceuticallyacceptable salts include, but are not limited to, mineral or organicacid salts of basic residues such as amines; alkali or organic salts ofacidic residues such as carboxylic acids; and the like. Thepharmaceutically acceptable salts include the conventional non-toxicsalts or the quaternary ammonium salts of the parent compound formed,for example, from non-toxic inorganic or organic acids. For example,such conventional non-toxic salts include those derived from inorganicacids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric,nitric and the like; and the salts prepared from organic acids such asacetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric,citric, ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic, glutamic,benzoic, salicylic, sulfanilic, 2-acetoxybenzoic, fumaric,toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isethionic,and the like.

The pharmaceutically acceptable salts of the present invention can besynthesized from the parent compound which contains a basic or acidicmoiety by conventional chemical methods. Generally, such salts can beprepared by reacting the free acid or base forms of these compounds witha stoichiometric amount of the appropriate base or acid in water or inan organic solvent, or in a mixture of the two; generally, nonaqueousmedia like ether, ethyl acetate, ethanol, isopropanol, or acetonitrileare preferred. Lists of suitable salts are found in Remington'sPharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa.,1985, p. 1418, the disclosure of which is hereby incorporated byreference. Said references are incorporated herein by reference.

In addition, the compound of formula I is, subsequent to itspreparation, preferably isolated and purified to obtain a compositioncontaining an amount by weight equal to or greater than 99% formula Icompound (“substantially pure” compound I), which is then used orformulated as described herein. Such “substantially pure” compounds offormula I are also contemplated herein as part of the present invention.

All stereoisomers of the compound of the instant invention arecontemplated, either in admixture or in pure or substantially pure form.The compound of the present invention can have asymmetric centers at anyof the carbon atoms including any one of the R substituents and/orexhibit polymorphism. Consequently, the compound of formula I can existin enantiomeric, or diastereomeric forms, or in mixtures thereof. Theprocesses for preparation can utilize racemates, enantiomers, ordiastereomers as starting materials. When diastereomeric or enantiomericproducts are prepared, they can be separated by conventional methods forexample, chromatographic or fractional crystallization.

“Stable compound” and “stable structure” are meant to indicate acompound that is sufficiently robust to survive isolation to a usefuldegree of purity from a reaction mixture, and formulation into anefficacious therapeutic agent. The present invention is intended toembody stable compounds.

“Therapeutically effective amount” is intended to include an amount of acompound of the present invention alone or an amount of the combinationof compounds claimed or an amount of a compound of the present inventionin combination with other active ingredients effective to inhibit MIP-1αor effective to treat or prevent inflammatory disorders.

As used herein, “treating” or “treatment” cover the treatment of adisease-state in a mammal, particularly in a human, and include: (a)preventing the disease-state from occurring in a mammal, in particular,when such mammal is predisposed to the disease-state but has not yetbeen diagnosed as having it; (b) inhibiting the disease-state, i.e.,arresting it development; and/or (c) relieving the disease-state, i.e.,causing regression of the disease state.

SYNTHESIS

The compound of formula I was prepared as shown in the followingExample, reaction scheme and descriptions thereof, as well as relevantliterature procedures that may be used by one skilled in the art.Exemplary reagents and procedures for these reactions appearhereinafter.

Step 1: tert-butyl 3-methyl-4-oxopiperidine-1-carboxylate

A mixture of 1-benzyl-3-methylpiperidin-4-one (11.4 g, 56.1 mmol),Boc-anhydride (14.32 mL, 61.7 mmol), and 10% palladium on carbon (0.597g, 5.61 mmol) in ethanol (100 mL) was degassed under vacuum andnitrogen, then hydrogenated at 50 psi for 4 hours at room temperature.The catalyst was removed by filtration, rinsed with methanol, and thecombined filtrate and rinses were concentrated in vacuo. The residue waspurified over a 330 g silica gel column, eluting at 100 mL/min with anethyl acetate/hexanes gradient to yield tert-butyl3-methyl-4-oxopiperidine-1-carboxylate (11.66 g, 54.7 mmol, 97% yield)as an oil, which solidified upon standing. ¹H NMR (CDCl₃, 400 MHz) δ4.15 (m, 2H), 3.22 (m, 1H), 2.79 (br m, 1H), 2.52-2.38 (m, 3H), 1.46 (s,9H), 1.01 (d, J=6.8 Hz, 3H); m/z=158.2 (M-tBu)⁺.

Step 2: tert-butyl4-(4-chlorophenyl)-3-methyl-5,6-dihydropyridine-1(2H)-carboxylate

A solution of 1-bromo-4-chlorobenzene (21.87 g, 114 mmol) in anhydrousTHF (250 mL) was cooled to −78° C. and treated dropwise withn-butyllithium (43.5 mL, 109 mmol, 1.6 M in hexanes). The mixture wasstirred at −78° C. for 45 minutes, during which time a precipitate wasobserved, then the slurry was treated dropwise with a solution oftert-butyl 3-methyl-4-oxopiperidine-1-carboxylate (11.6 g, 54.4 mmol) inanhydrous THF (50 mL). The reaction was stirred at −78° C. for 2 hours,then allowed to slowly warm to −20° C. and quenched with saturatedammonium chloride. The layers were separated, and the organic phase wasconcentrated in vacuo. The aqueous phase was extracted once with ethylacetate (300 mL), and the organic phase was combined with the residuefrom the original organic phase. The mixture was washed 3 times withwater, and once with brine, then dried over sodium sulfate andconcentrated in vacuo. The residue was digested in boiling hexanes (200mL) for 1 hour, and then the mixture was cooled to room temperature. Thesolids were collected by filtration, rinsed with a small amount of hothexanes, and dried under vacuum to yield tert-butyl4-(4-chlorophenyl)-4-hydroxy-3-methylpiperidine-1-carboxylate (13.96 g,42.8 mmol, 79% yield) as a powder. m/z=252.2 (M-tBuO)⁺.

In a 250 mL round-bottom flask, tert-butyl4-(4-chlorophenyl)-4-hydroxy-3-methylpiperidine-1-carboxylate (4.3 g,13.2 mmol) was treated with concentrated HCl (25 mL, 300 mmol). Themixture was stirred at room temperature until the vigorous foamingceased and a heterogeneous free-stirring mixture was observed. Themixture was heated at reflux for 19 hours, during which a clear solutionwas observed.

The reaction mixture was concentrated in vacuo, and the residue wasconcentrated twice from toluene to remove residual HCl. The residue wassuspended in THF (50 mL), and added Boc-anhydride (3.00 mL, 12.9 mmol)followed by triethylamine (5.52 mL, 40 mmol). The reaction was stirredat room temperature for 2 hours. The mixture was then concentrated invacuo, and the residue was taken up in ethyl acetate (150 mL). Theheterogeneous mixture was washed with 1 N HCl (3×), once with water, andonce with brine, then dried over sodium sulfate and concentrated invacuo to yield tert-butyl4-(4-chlorophenyl)-3-methyl-5,6-dihydropyridine-1(2H)-carboxylate (4.3g) as an oil. ¹H NMR (400 MHz, MeOD₄) δ 7.34 (m, 2H), 7.14 (d, J=8.3 Hz,2H), 3.87 (br s, 2H), 3.58 (br s, 2H), 2.33 (m, 2H), (1.58 (s, 3H), 1.48(s, 9H); m/z (ESI⁺)=252.1 (M-tert-Bu)⁺.

Step 3: 3S,4S)-tert-butyl4-(4-chlorophenyl)-3,4-dihydroxy-3-methylpiperidine-1-carboxylate

A solution of tert-butyl4-(4-chlorophenyl)-3-methyl-5,6-dihydropyridine-1(2H)-carboxylate (2.2g, 7.15 mmol) in 50% tert-butanol/water (50 mL) was cooled to 5° C., andtreated with methanesulfonamide (0.680 g, 7.15 mmol) and AD-Mix alpha(11 g, 7.15 mmol). The resulting suspension was stirred at 5° C. for 4hours, then allowed to slowly come to room temperature and stirred for 3days. The mixture was then cooled to 5° C. and treated with solid sodiumsulfite, and the mixture was stirred for 1 hour, during which time aclear solution was observed. The mixture was extracted with ethylacetate (3×), then the combined organic phases were washed twice withwater, and once with brine, dried over sodium sulfate, and concentratedin vacuo. The residue was purified via MPLC over a 120 g silica gelcolumn, eluting at 85 mL/min with 25% then 30% then 35% ethylacetate/hexanes to yield (3S,4S)-tert-butyl4-(4-chlorophenyl)-3,4-dihydroxy-3-methylpiperidine-1-carboxylate (2.1g, 5.53 mmol, 77% yield) as an oil. ¹H NMR (400 MHz, MeOD₄) δ 7.53 (d,J=8.8 Hz, 2H), 7.30 (d, J=8.8 Hz, 2H), 4.05 (br d, 1H), 3.73 (m, 1H),3.26 (m, 2H), 2.48 (ddd, J=12.7, 4.8 Hz, 1H), 1.65 (d, J=14.1 Hz, 1H),1.47 (s, 9H), 0.92 (s, 3H); m/z (ESI+)=342.1 (M+H)⁺, 286.1 (M-tert-Bu)⁺.

Step 4: (3S,4S)-4-(4-chlorophenyl)-3-methylpiperidine-3,4-diolHydrochloride

A solution of (3S,4S)-tert-butyl4-(4-chlorophenyl)-3,4-dihydroxy-3-methylpiperidine-1-carboxylate (2.08g, 6.08 mmol) in 4 M HCl in dioxane (30.4 mL, 122 mmol) was stirred atroom temperature for 45 minutes. The mixture was concentrated in vacuo,then concentrated from methylene chloride (3×) to remove residual HCl,to yield (3S,4S)-4-(4-chlorophenyl)-3-methylpiperidine-3,4-diolhydrochloride (1.7 g, 6.11 mmol, 100% yield) as a powder. ¹H NMR (400MHz, MeOD₄) δ 7.55 (d, J=8.8 Hz, 2H), 7.36 (d, J=8.8 Hz, 2H), 3.42 (m,1H), 3.29 (m, 1H), 3.05 (d, J=13.2 Hz, 1H), 2.71 (ddd, J=13.2, 4.8 Hz,1H), 1.97 (dt, J=14.5, 3.1 Hz, 1H), 1.08 (s, 3H); m/z (ES+)=242.1(M+H)⁺.

Step 5: tert-butyl(R)-1-((3S,4S)-4-(4-chlorophenyl)-3,4-dihydroxy-3-methylpiperidin-1-yl)-3-methyl-1-oxobutan-2-ylcarbamate

To a mixture of (3S,4S)-4-(4-chlorophenyl)-3-methylpiperidine-3,4-diol(91 g, 376 mmol), Boc-D-Valine (83.1 g, 382 mmol), HOBt (69.2 g, 452mmol), EDC (87 g, 452 mmol) in CH₂Cl₂ (800 mL) and DMF (80 mL) was addedDIPEA (132 mL, 753 mmol) at room temperature. The mixture was stirred atroom temperature overnight. The reaction was concentrated and theresidue partitioned between EtOAc and water. The EtOAc layer was washedwith 1N HCl, sat. aq. NaHCO₃, and brine to give the crude product (180g, ˜80% purity) which was used directly in the next transformation.

Step 6: (3S,4S)-4-(4-chlorophenyl)-3-methyl-1-D-valyl-3,4-piperidinediol

A solution of tert-butyl(R)-1-(3S,4S)-4-(4-chlorophenyl)-3,4-dihydroxy-3-methylpiperidin-1-yl)-3-methyl-1-oxobutan-2-ylcarbamate(0.150 kg, 0.34 mol) and HCl (408 mL, 1.02 mol, 2.5 M) in EtOH (18 mL)was stirred at 60° C. for 2 hours. The solvent was then removed to givea oil which was added EtOAc (1 L), water (1 L) and TEA (2 mol). The twolayer solution was stirred at room temperature for 2 h and the productprecipitated as a solid (70 g, 98% purity). An additionalrecrystallizion from MeOH enhanced purity to 99.8%. An additional 11 gwas recovered from mother liquors to provide(3S,4S)-4-(4-chlorophenyl)-3-methyl-1-D-valyl-3,4-piperidinediol (81 g,70% yield) as a solid. ¹H NMR (400 MHz, MeOD₄, rotameric) δ 7.54 (m,2H), 7.31 (m, 2H), 4.53 (m, 0.5H), 4.27 (dd, J=12.4, 1.2 Hz, 0.5H), 3.93(br d, 0.5H), 3.70 (t, J=5.2 Hz, 1H), 3.69 (m, 1.5H), 3.14 (m, 1H), 2.49(ddd, J=13.9, 4.8 Hz, 1H), 1.96-1.71 (m, 2H), 1.07-0.89 (m, 9H);m/z=341.12 (M+H)⁺.

Step 7: (R)-benzyl 3,3-difluorocyclopentanecarboxylate

(R)-benzyl 3-oxocyclopentanecarboxylate (62.8 g, 288 mmol) was dissolvedin dichloroethane (220 mL) and cooled to 10° C. with an ice-bath. Tothis mixture was added (diethylamino)sulfur trifluoride (86 mL, 647mmol) and the reaction temperature was kept below rt during theaddition. After warming to room temperature, the solution was heated at40° C. for 12 hr and then stirred at room temperature for 12 hr to givea black solution. The resulting solution was cooled with dry ice/MeOHand quenched with MeOH (30 mL). The mixture was then poured into a 4 Lbeaker cooled with dry ice/MeOH and an aqueous NaHCO₃+Na₂CO₃ solutionwas added slowly to adjust to pH 5-7. The product was extracted with DCMand the organic layer was concentrated to give a liquid (>80 g). Thecrude product was purified via column chromatography EtOAc/hexanes(0-10% gradient, 15 min) to furnish 44 g of (R)-benzyl3,3-difluorocyclopentanecarboxylate as a liquid. ¹H NMR (CDCl₃, 400 MHz)δ 7.23 (m, 5H), 3.05 (m, 1H), 2.34-2.45 (m, 2H), 2.01-2.16 (m, 4H).m/z=263.2 (M+Na)⁺.

Step 8: (R)-3,3-difluorocyclopentanecarboxylic acid

(R)-benzyl 3,3-difluorocyclopentanecarboxylate (13 g, 54.1 mmol) wasdissolved in MeOH (200 mL) at room temperature with stirring then 0.5 MLiOH (216 mL, 108 mmol) was added. After stirring for 2 h, water wasadded to the reaction mixture and concentrated to remove volatilesolvents. The aqueous layer was rinsed with Et₂O (2×) then acidified topH 3 with 1N HCl. The aqueous layer was extracted with methylenechloride (2×) and the organic layers were combined, dried (Na₂SO₄), andconcentrated to give (R)-3,3-difluorocyclopentanecarboxylic acid (6.58g, 43.8 mmol, 81% yield) as an oil. ¹H NMR (MeOD₃, 400 MHz) δ 3.03 (m,1H), 2.33 (m, 2H), 1.95-2.20 (m, 4H); m/z=149.2 (M−H).

Step 9:(R)—N—((R)-1-((3S,4S)-4-(4-chlorophenyl)-3,4-dihydroxy-3-methylpiperidin-1-yl)-3-methyl-1-oxobutan-2-yl)-3,3-difluorocyclopentaneCarboxamide

To a mixture of(R)-2-amino-1-((3S,4S)-4-(4-chlorophenyl)-3,4-dihydroxy-3-methylpiperidin-1-yl)-3-methylbutan-1-one(22.49 g, 66.0 mmol), (R)-3,3-difluorocyclopentanecarboxylic acid (10.21g, 68.0 mmol) in CH₂Cl₂ (300 mL) and DMF (75 mL) were added HOBT (12.50g, 82 mmol), EDC (15.65 g, 82 mmol) and Hunig's base (23.76 mL, 136mmol). The reaction was stirred for 20 h at rt then concentrated toremove the CH₂Cl₂. Ethyl acetate was added and the organic layer waswashed twice with 1N HCl, twice with saturated sodium carbonate andthree times with water. The organic layer was dried over sodium sulfateand concentrated to give a glass solid which was crystallized from CH₃CNto give(R)—N—((R)-1-((3S,4S)-4-(4-chlorophenyl)-3,4-dihydroxy-3-methylpiperidin-1-yl)-3-methyl-1-oxobutan-2-yl)-3,3-difluorocyclopentanecarboxamide(31.03 g, 65.6 mmol, 99% yield).

¹H NMR (500 MHz, DMSO-d6, rotameric) δ 8.11 (m 1H), 7.55 (m, 2H), 7.34(app t, 2H), 5.29 (d, J=6.9 Hz, 1H), 4.76 (m, 0.5H), 4.68-4.58 (m,1.5H), 4.43 (br m, 0.5H), 4.09 (br d, 0.5H), 3.97 (br d, 0.5H), 3.50 (m,1H), 3.35 (m, 3H), 3.00 (m, 2H), 2.31 (m, 0.5H), 2.32-1.94 (m, 6.5H),1.77 (m, 1H), 1.65 (m, 1H), 0.92-0.83 (m, 6H), 0.74 (m, 3H); m/z=473.2(M+H)

Utility

In general, the compound of formula (I) has been shown to be a modulatorof chemokine receptor activity. By displaying activity as a modulator ofchemokine receptor activity, the compound of formula (I) is expected tobe useful in the treatment of human diseases associated with chemokinesand their cognate receptors.

Comparative Pharmacological Characteristics

Assays and data comparing the pharmacological and physiochemicalcharacteristics of Compound I and compounds found in US2007/0208056A1(corresponding to WO 2007/092681) is presented below.

The compound of the present invention (Compound I) has been compared toother compounds that have been found to be useful inhibitors of CCR-1activity, and found to be especially advantageous. For example, thesurprising advantage over the other compounds is shown below in Tables 1and 2.

Human CCR1THP-1 Binding Assay

For radioligand competition studies, a final concentration of 1×10⁵THP-1 monocytic leukemia cells are combined with 100 μg of LS WGA PSbeads (Amersham, Cat.#: RPNQ 0260) in 40 μL of assay buffer (RPMI 1640without phenol red, 50 mM HEPES, 5 mM MgCl₂, 1 mM CaCl₂, 0.1% BSA). TheTHP-1 cell/bead mixture was added to each well of a 384-well assay plate(PerkinElmer, Cat. #:6007899) containing test compound in 3-fold serialdilution, with final concentrations ranging from 8 μM to 0.14 nM. Afinal concentration of 0.1 nM [¹²⁵I]MIP-1α (PerkinElmer, Cat. # NEX298)in 20 μL assay buffer was added to the reaction. Unlabeled MIP-1α wasadded in excess to some wells to determine non-specific binding. Sealedassay plates were incubated at room temperature for 12 h then analyzedby LEADseeker™.

The competition data of the test compound over a range of concentrationsis plotted as percentage inhibition of radioligand specifically bound inthe absence of test compound (percent of total signal). After correctingfor non-specific binding, IC₅₀ values are determined. The IC₅₀ value isdefined as the concentration of test compound needed to reduce[¹²⁵I]-MIP-1α specific binding by 50% and is calculated using the fourparameter logistic equation to fit the normalized data. The K_(i) valuesare determined by application of the Cheng-Prusoff equation to the IC₅₀values, where K_(i)=IC₅₀/(1+ligand concentration/K_(d)). The Kd of[¹²⁵I]-MIP-1α in THP-1 cells is 0.1 nM. Each experiment was run induplicate.

hERG Patch Clamp Assay

Whole-cell patch-clamp was used to directly measure hERG tail currentsin HEK-293 cells stably expressing the cloned hERG potassium channel αsubunit. Effects of compounds were calculated by measuring inhibition ofpeak tail current. Experiments were carried out using an aqueous bufferwith pH 7.4 at room temperature. There was no protein in the assaybuffer. The test concentrations reported are nominal free drug levels.Data is reported as percent inhibition at a fixed concentration.

Sodium and L-Type Calcium Channel Assays

Whole-cell patch-clamp was used to directly measure inward sodiumcurrents in HEK-293 cells expressing the human cardiac sodium channel,SCNSA. After reach steady-state effect in the presence of drug,rate-dependence was assessed by stimulation at frequencies of 1 Hz and 4Hz. Experiments were carried out using an aqueous buffer at pH 7.4 andat room temperature. There is no protein in the buffer and drugconcentrations reported are nominal free drug levels. Test article wasevaluated up to 10 μM (protein-free buffer). Rate-dependence ofinhibition was assessed by stimulation at frequencies of 1 Hz and 4 Hz.Data is reported as percent inhibition at a fixed concentration.

In addition to the potential for interaction with the L-type calciumchannel, whole-cell patch-clamp was used to directly measure inwardcalcium currents in HEK-293 cells stably expressing the cloned humancardiac L-type Ca channel (α1C) and its β subunit. Effects of compoundswere calculated by measuring inhibition of peak current. Experimentswere carried out using an aqueous buffer at pH 7.4 and at roomtemperature. There is no protein in the buffer and drug concentrationsreported are nominal free drug levels. Data is reported as percentinhibition at a fixed concentration.

Electrocardiography in Anesthetized Rabbits

A dose-response study of test compound was conducted in anesthetizedrabbits to assess the cardiac electrophysiologic profile established inthe cellular ion channel assays.

Experiments were performed in propofol-fentanyl anesthetizedclosed-chest male rabbits. A body surface electrocardiogram (ECG) and anintra-cardiac His-bundle electrogram were continuously monitored andrecorded during the studies, using a PoNeMah system and a Pruckaelectrophysiological recording system, respectively. Test compound wasprepared on the day of study in a vehicle of PEG400:ethanol:water(1:1:1) at a dosing concentration of 30 mg/mL. Test compound (n=3) orvehicle (n=3) was given intravenously over 5 minutes via an infusionpump at incremental doses of 3, 10 and 30 mg/kg. The interval betweendoses was 10 minutes, allowing for a 5-minute test agent infusion and a5-minute rest period. Blood was sampled at baseline (pre-drug infusion)and immediately at the end of each infusion. For the 30 mg/kg dose,additional blood samples were taken at 10, 20 and 30 minutes after theend of infusion.

PR interval, QRS duration and QT interval were averaged from a 1 minuteECG recording period at time of blood sampling. QT interval wascorrected for heart rate effects using both Fridericia (QTcf) and Vander Water (QTcv) formulae. AH and HV intervals, representing A-V nodeconduction and His-Purkinje conduction, respectively, were assessed bymanual measurements from the His-bundle electrogram. Data were expressedas percent change from pre-drug baseline (mean±SEM) for PR, QRS, AH andHV intervals as well as delta change from the pre-drug infusion baseline(mean±SEM) for QTc intervals. Changes of 10% in PR, QRS, and AHintervals, and 20% in HV interval, as well as >10 ms in QTc interval areconsidered significant based on experience with the model.

As shown below in Table 1, the in vivo data demonstrates the superiorsafety profile of Compound I. In particular, while showing a lower invitro CCR1 Ki as compared to the other compounds, Compound I also had aNo Observed Effect Level (NOEL) in the rabbit of 30 mg/kg. While thecompound of Example 491 had a similar NOEL, it had a higher absolute QTprolongation as well as a lower free fraction of circulating drugrelative to Compound I.

TABLE 1 In Vitro and In Vivo Cardiovascular Safety Profile AssayCompound I EX# 491* EX# 572* CCR1 Ki (nM) 1.6 2.1 1.5 hERG channel, 22%@ 30 μM 27% @ 30 μM 32% @ 30 μM % inh Na channel, % inh 21% @ 10 μM 13%@ 10 μM 25% @ 10 μM Ca channel, % inh 29% @ 30 μM 22% @ 10 μM 19% @ 30μM Protein binding 68% 95% 84% Human (rabbit 54%) (rabbit 94%) (rabbit87%) Rabbit In Vivo EP Effect QTcf, delta No effect +17 ms +22 ms QTeffect No effect up to 30 mg/kg 10 mg/kg Dose 30 mg/kg 138.5/5.5 μM48.6/6.3 μM Cmax: Total 96/52 μM drug/Free drug NOEL dose, 30 mg/kg 10mg/kg Not identified Cmax: Total 96/52 μM 77/3.1 μM <10 mg/kg drug/Freedrug <48.6/6.3 μM *Examples from US2007/0208056 as shown below: Ex. 491 

Ex. 572 

PXR Transactivation Assay

The cell culture medium used is DMEM. Lipofectamine 2000, PBS,heat-inactivated fetal bovine serum (FBS), trypsin-EDTA (0.25%), andpenicillin-streptomycin were purchased from GIBCO/Invitrogen (Carlsbad,Calif.). Charcoal/dextran treated fetal bovine serum (FBS) was purchasedfrom Hyclone (Logan, Utah). HepG2 cells were obtained from ATCC(Manassas, Va.). Human PXR-pcDNA3 and luciferase reporter containingCYP3A4 promoter, CYP3A-Luc, were generated at Bristol-Myers Squibb.White tissue culture (TC)-surface 384-well plates were purchased fromPerkin Elmer (Boston, Mass.). Luciferase substrate (Steady-Glo) waspurchased from Promega (Madison, Wis.). Control compounds rifampicin,mifepristone, and sulfinpyrazone were purchased from Sigma (St. Louis,Mo.).

Culture of HepG2 cells is performed in T175 flasks using DMEM containing10% FBS. The transfection mixture contains 1 μg/mL of PXR-pcDNA3 plasmidDNA, 20 μg/ml of Cyp3A-Luc plasmid DNA, 90 μL/mL of Lipofectamine 2000,and serum-free medium. After incubating at room temperature for 20minutes, the transfection mixture (1 ml per flask) is applied to thecells in fresh medium (20 mL per flask), and flasks incubated at 37° C.(5% CO₂) overnight.

Cells in each flask are washed with PBS and 2 mL of Trypsin-EDTA (0.25%)is added and incubated for five minutes at 37° C., 5% CO₂. The flasksare then tapped vigorously to break up cell aggregates. After theaddition of 8 mL of DMEM containing 5% charcoal/dextran-treated FBS, theentire mixture is transferred to conical tubes. Cells are thencentrifuged at 1000 rpm for 5 minutes. Cell pellets are resuspended to afinal count of ˜7×10⁶ cells/mL in freezing media (DMEM containing 20%serum and 10% DMSO). The cell suspension is aliquoted into 15-mLpolypropylene tubes, 5 mL per tube. Cells are slowly frozen by placingin a Styrofoam-insulated container at −80° C. overnight. Vials aretransferred to an Ultracold (−140° C.) freezer after 24 hours forlong-term storage.

Vials of cryopreserved cells are thawed rapidly in a warm water bath forfive minutes. Cells are pooled and diluted to 50 mL in a 50-mL conicalvial. The thawed cells are centrifuged at 1500 rpm for 5 minutes tocollect the cells and the supernatant discarded. Cells are thenresuspended in fresh Media II (DMEM containing 5%charcoal/dextran-treated FBS, 1% Penicillin/Streptomycin, 100 μMNon-essential Amino Acids, 1 mM Sodium Pyruvate, and 2 mM L-Glutamine),counted using the Guava Cell Counter, and diluted to 1.6×10⁵ cells/ml inthe same media.

Fifty microliters of cell mixture is added to wells in columns 1-23 ofwhite tissue-culture treated 384-well plates containing 0.25 μL of testcompound dissolved in 100% DMSO. Fifty microliters of Media II is addedto wells in column 24. The plates are incubated at 37° C. (5% CO₂) for24 hours, then 5 μL of Alamar Blue reagent (Trek Diagnostics, Cat#00-100) is added to each well. Plates are then incubated an additionaltwo hours at 37° C., 5% CO₂ and then one hour at room temperature.Fluorescence is read at Ex525/Em598. After the fluorescence is measured,25 μL of luciferase substrate (Steady-Glo, Promega) is added to eachwell. The plates are incubated for fifteen minutes at room temperature,after which the luminescence is read on a PheraStar (BMG Labtech) platereader.

Rifampicin (10 μM), a well-known agonist of PXR, is included in eachplate as an internal standard and positive control. The data is thenexpressed as percent control (% CTRL), where the control signal is thesignal from the 10 μM rifampicin and the blank signal is that from theDMSO vehicle.

% CTRL=((Compound signal−Blank signal)/(Control signal−Blanksignal))*100

Compounds are tested at ten concentrations (2.5 nM-50 μM, 1:3 serialdilution). Assay results are reported as EC₅₀, the concentration ofcompound at which 50% of the maximal response is observed, and asYMAXOBS, the maximal response (highest percent CTRL) observed for thatcompound. The EC₅₀ is defined as the concentration corresponding to halfof the maximal response derived from the fitted 20-point curve asdetermined using a four-parameter logistic regression model.Additionally, compounds may also be reported as EC₂₀ or EC₆₀.

Data Analysis for HepG2 Cytotoxicity Assay

Compounds are tested at ten concentrations (2.5 nM-50 μM, 1:3 serialdilution). Assay results are reported as IC₅₀, defined as theconcentration corresponding to 50 percent inhibition as derived from thefitted 20-point curve determined using a four-parameter logisticregression model.

In Vitro Metabolism Assays Assay Conditions A:

Test compound is received as a 3.5 mM stock solution in 100 percentDMSO. Compound is diluted to create a 50 μM acetonitrile (ACN) solutioncontaining 1.4% DMSO, which is then used as a 100× stock for incubationwith microsomes. Each compound is tested in duplicate separately in eachof three species in the Metabolic Stability-Human, Rat, and Mouse assaysuite or as individual species in the Metabolic Stability-Dog orMetabolic Stability-Monkey suites. Compound, NADPH and liver microsomesolutions are combined for incubation in three steps:

152 μL of liver microsome suspension, protein concentration of 1.1 mg/mLin 100 mM NaP_(i), pH 7.4, 6.6 mM MgCl₂ buffer, is pre-warmed at 37° C.

-   1) 1.7 μL of 50 μM compound (98.6% ACN, 1.4% DMSO) is added to the    same tube and pre-incubated at 37° C. for 5 minutes.-   2) The reaction is initiated by the addition of 17 μL of pre-warmed    10 mM NADPH solution in 100 mM NaP_(i), pH 7.4.-   3) Reaction components are mixed well, and 75 μL are immediately    transferred into 150 μl quench/stop solution (zero-time point,    T_(o)). Reactions are incubated at 37° C. for 10 minutes and then an    additional 75 μL aliquot is transferred into 150 μL quench solution.    Acetonitrile containing 100 μM DMN (a UV standard for injection    quality control), is used as the quench solution to terminate    metabolic reactions.-   4) Quenched mixtures are centrifuged at 1500 rpm (˜500×g) in an    Allegra X-12 centrifuge, SX4750 rotor (Beckman Coulter Inc.,    Fullerton, Calif.) for fifteen minutes to pellet denatured    microsomes. A volume of 90 μL of supernatant extract, containing the    mixture of parent compound and its metabolites, is then transferred    to a separate 96-well plate for UV-LC/MS-MS analysis to determine    the percent of parent compound that is remaining in the mixture.

Metabolic Stability Assay-Reaction Components Final Concentration in theReaction Components Metabolic Stability Assay Compound (Substrate) 0.5μM NaPi Buffer, pH 7.4 100 mM DMSO 0.014% Acetonitrile 0.986% Microsomes(human, rat, mouse) 1 mg/mL (BD/Gentest) protein NADPH 1.0 mM MgCl₂ 6.66mM 37° C. Incubation time 0 minutes and 10 minutes Quench/Stop Solution(ACN + 100 μM 150 μL DMN) Sample of Reaction 75 μL Sedimentation ofDenatured Microsomes 15 minutes UV-LC/MS analysis of supernatant 0.17 μM

Assay Conditions B:

Test compound is received as 20 mM in DMSO. Compound is diluted tocreate a 300 μM acetonitrile (ACN) solution containing 1.5% DMSO, whichis then used as a 100× stock for incubation with microsomes. Eachcompound is tested in duplicate separately in each of three species inthe Metabolic Stability-Human, Rat, Mouse assay suite or individualspecies in the Metabolic Stability-Dog or Metabolic Stability-Monkeysuites. Compound, NADPH and liver microsome solutions are combined forincubation in three steps:

-   1. 450 μL of liver microsome suspension, protein concentration of 1    mg/mL in 100 mM NaP_(i), pH 7.4, 6.6 mM MgCl₂ buffer, are pre-warmed    at 37° C.-   2. 5 μA of 300 μM compound (98.5% CAN, 1.5% DMSO) is added to the    same tube.-   3. The reaction is initiated by the addition of 50 μL of pre-warmed    5 mM NADPH solution in 100 mM NaP_(i), pH 7.4.

Reaction components are mixed well and 150 μL removed to quench/stopsolution immediately for the zero-minute time point. Reactions areincubated at 37° C. for 10 minutes and then an additional 150 μL removedfrom incubation. Aliquots removed are combined with 300 μL ACN whichcontains 100 μM DMN as a UV standard for detection.

Final Concentration in the Reaction Components Metabolic Stability AssayCompound (Substrate) 3 μM NaPi Buffer, pH 7.4 100 mM DMSO 0.015% ACN0.985% Microsomes (human, rat, mouse) 1 mg/ml (BD/Gentest) protein NADPH0.5 mM MgCl₂ 6.66 mM 37° C. Incubation time 0 minutes and 10 minutesQuench/Stop Solution (ACN + 100 μM 300 μL DMN) Sample of Reaction 150 μLSedimentation of Denatured Microsomes 15 minutes UV-LC/MS analysis ofsupernatant- 1.0 μM

Quenched mixtures are centrifuged at 1500 rpm (˜500×g) in an AllegraX-12 centrifuge, SX4750 rotor (Beckman Coulter Inc., Fullerton, Calif.)for fifteen minutes to pellet denatured microsomes. A volume of 110 μlof supernatant extract, containing the mixture of parent compound andits metabolites, is then transferred to a separate 96-well plate forUV-LC/MS-MS analysis to determine the percent of parent compound that isremaining in the mixture.

As shown below in Table 2, Compound I can also be positivelydifferentiated through 2 critical parameters, PXR transactivation andhuman liver microsome stability.

PXR transactivation predicts potential drug-drug interactions. If acompound activates this receptor, other drugs may be metabolized quickerthan normal leading to lower drug levels and reduced efficacy. This isclearly an unwanted characteristic in a compound that is beingconsidered for development.

The in vitro screen indicates that Compound I is not a significantactivator of this receptor relative to all but one of the compoundslisted.

Finally, the compounds were tested in the human liver microsomestability assay which is a good predictor of in vivo clearance of acompound. For development purposes, compounds with less than 70% weredropped from further consideration.

Thus, the combination of the low risk of PXR transactivation along withthe 100% of compound remaining in the human liver microsome metabolismassay shows that Compound I has superior pharmacological characteristicswhen compared with other known and structurally similar CCR-1antagonists.

TABLE 2 PXR/Cyp 3A4 Induction potential and Metabolic Stability - InVitro: Human Liver Microsomal Metabolism CCR1 K_(i) PXR EC₅₀ % remainingExample #* (nM) (μM) (Assay Conditions) Compound I 1.6 >50 96% (B) #13563.7 1.83 100% (A)/100% (B) #1083 2.6  1.9 (EC₂₀) 13% (A) #460 0.7 0.5347% (A)/16% (B) #466 2.6 1.12 (EC₆₀) 67% (A)/33% (B) #850 0.5 10.2(EC₆₀) 99% (A) #897 2.1 >50 (EC₆₀) 59% (A) *Examples from US2007/0208056as shown below: Ex. 1356 

Ex. 460 

Ex. 466 

Ex. 850 

Ex. 897 

Ex. 1083 

Single-Dose Pharmacokinetics in Rats

Male Sprague-Dawley rats were dosed with compound (N=3 animals) in asolution orally or by IV infusion. Serial blood samples were collectedfor all studies from the jugular vein at various time points post dose(0.17 (or 0.25 for oral), 0.5, 0.75, 1, 2, 4, 6, 8 and 24 h, forexample). Plasma samples were analyzed by LC-MS/MS.

Additional studies evaluating suspensions of crystalline drug substancein aqueous medium were performed as described above.

Single-Dose Pharmacokinetics in Dogs

Male beagle dogs were dosed with compound (N=3 animals) in a solutionorally or by IV infusion. Serial blood samples were collected for allstudies at various time points post dose (0.17 (or 0.25 for oral), 0.5,0.75, 1, 2, 4, 6, 8 and 24 h, for example). Plasma samples were analyzedby LC-MS/MS.

As shown below in Table 3, the in vivo pharmacokinetic profiledemonstrates the superiority of Compound I in two different species. Inparticular, either solution or aqueous crystalline suspension in ratsdemonstrated a higher level of exposure relative to other compounds inthe table. The advantage of this profile is the predicted superiorperformance of solid dosage formulations in the clinic. Additionally, indogs, compound I shows a superior profile with respect to in vivoclearance (CL). Relative to the compared compounds, an 8-13-folddecrease is demonstrated. The combined superior performance of thesuspension formulations and decreased clearance positivelydifferentiates Compound 1.

TABLE 3 Pharmacokinetic Profile of Compound 1 Compound I EX# 491* EX#572* EX# 460 EX# 463 Species rat rat rat rat rat Rat oral 43% 23% 37% 8%9% bioavailability when dosed as solution Relative bio- 84% 0% >90% NotNot availability tested tested when dosed as a crystalline suspension CL26 67 29 10 75 (mL/min/kg) Species dog dog dog dog oral bio- 82 100 72Not Not availability tested tested when dosed as solution CL 0.6 7.9 4.9(mL/min/kg) *Examples from US2007/0208056 as shown below: Ex. 491 

Ex. 572 

Ex. 460 

Ex. 463 

Mammalian chemokine receptors provide a target for interfering with orpromoting immune cell function in a mammal, such as a human. Compoundsthat inhibit or promote chemokine receptor function are particularlyuseful for modulating immune cell function for therapeutic purposes.

Accordingly, the present invention is directed to a compound of formula(I) which is believed to be useful in the prevention and/or treatment ofa wide variety of inflammatory, infectious, and immunoregulatorydisorders and diseases, including asthma and allergic diseases,infection by pathogenic microbes (which, by definition, includesviruses), as well as autoimmune pathologies such as the rheumatoidarthritis and arthrosclerosis.

For example, the instant compound which inhibits one or more functionsof a mammalian chemokine receptor (e.g., a human chemokine receptor) maybe administered to inhibit (i.e., reduce or prevent) inflammation orinfectious disease. As a result, one or more inflammatory process, suchas leukocyte emigration, adhesion, chemotaxis, exocytosis (e.g., ofenzymes, histamine) or inflammatory mediator release, is inhibited.

Similarly, the instant compound which promotes one or more functions ofthe mammalian chemokine receptor (e.g., a human chemokine) asadministered to stimulate (induce or enhance) an immune or inflammatoryresponse, such as leukocyte emigration, adhesion, chemotaxis, exocytosis(e.g., of enzymes, histamine) or inflammatory mediator release,resulting in the beneficial stimulation of inflammatory processes. Forexample, eosinophils can be recruited to combat parasitic infections. Inaddition, treatment of the aforementioned inflammatory, allergic andautoimmune diseases can also be contemplated for the instant compoundwhich promotes one or more functions of the mammalian chemokine receptorif one contemplates the delivery of sufficient compound to cause theloss of receptor expression on cells through the induction of chemokinereceptor internalization or the delivery of compound in a manner thatresults in the misdirection of the migration of cells.

In addition to primates, such as humans, a variety of other mammals canbe treated according to the method of the present invention. Forinstance, mammals, including but not limited to, cows, sheep, goats,horses, dogs, cats, guinea pigs, rats or other bovine, ovine, equine,canine, feline, rodent or murine species can be treated. However, themethod can also be practiced in other species, such as avian species.The subject treated in the methods above is a mammal, male or female, inwhom modulation of chemokine receptor activity is desired. “Modulation”as used herein is intended to encompass antagonism, agonism, partialantagonism and/or partial agonism.

Diseases or conditions of human or other species which can be treatedwith inhibitors of chemokine receptor function, include, but are notlimited to: inflammatory or allergic diseases and conditions, includingrespiratory allergic diseases such as asthma, allergic rhinitis,hypersensitivity lung diseases, hypersensitivity pneumonitis,eosinophilic cellulitis (e.g., Well's syndrome), eosinophilic pneumonias(e.g., Loeffler's syndrome, chronic eosinophilic pneumonia),eosinophilic fasciitis (e.g., Shulman's syndrome), delayed-typehypersensitivity, interstitial lung diseases (ILD) (e.g., idiopathicpulmonary fibrosis, or ILD associated with rheumatoid arthritis,systemic lupus erythematosus, ankylosing spondylitis, systemicsclerosis, Sjogren's syndrome, polymyositis or dermatomyositis);systemic anaphylaxis or hypersensitivity responses, drug allergies(e.g., to penicillin, cephalosporins), eosinophilia-myalgia syndrome dueto the ingestion of contaminated tryptophan, insect sting allergies;autoimmune diseases, such as rheumatoid arthritis, psoriatic arthritis,multiple sclerosis, systemic lupus erythematosus, myasthenia gravis,juvenile onset diabetes; glomerulonephritis, autoimmune thyroiditis,Behcet's disease; graft rejection (e.g., in transplantation), includingallograft rejection or graft-versus-host disease; inflammatory boweldiseases, such as Crohn's disease and ulcerative colitis;spondyloarthropathies; scleroderma; psoriasis (including T-cell mediatedpsoriasis) and inflammatory dermatoses such as an dermatitis, eczema,atopic dermatitis, allergic contact dermatitis, urticaria; vasculitis(e.g., necrotizing, cutaneous, and hypersensitivity vasculitis);eosinophilic myositis, eosinophilic fasciitis; cancers with leukocyteinfiltration of the skin or organs. Other diseases or conditions inwhich undesirable inflammatory responses are to be inhibited can betreated, including, but not limited to, reperfusion injury,arthrosclerosis, certain hematological malignancies, cytokine-inducedtoxicity (e.g., septic shock, endotoxic shock), polymyositis,dermatomyositis. Infectious diseases or conditions of human or otherspecies which can be treated with inhibitors of chemokine receptorfunction, include, but are not limited to, HIV.

Diseases or conditions of humans or other species which can be treatedwith promoters of chemokine receptor function, include, but are notlimited to: immunosuppression, such as that in individuals withimmunodeficiency syndromes such as AIDS or other viral infections,individuals undergoing radiation therapy, chemotherapy, therapy forautoimmune disease or drug therapy (e.g., corticosteroid therapy), whichcauses immunosuppression; immunosuppression due to congenital deficiencyin receptor function or other causes; and infections diseases, such asparasitic diseases, including, but not limited to helminth infections,such as nematodes (round worms); (Trichuriasis, Enterobiasis,Ascariasis, Hookworm, Strongyloidiasis, Trichinosis, filariasis);trematodes (flukes) (Schistosomiasis, Clonorchiasis), cestodes (tapeworms) (Echinococcosis, Taeniasis saginata, Cysticercosis); visceralworms, visceral larva migraines (e.g., Toxocara), eosinophilicgastroenteritis (e.g., Anisaki sp., Phocanema sp.), cutaneous larvamigraines (Ancylostona braziliense, Ancylostoma caninum). The compoundsof the present invention are accordingly useful in the prevention andtreatment of a wide variety of inflammatory, infectious andimmunoregulatory disorders and diseases.

In addition, treatment of the aforementioned inflammatory, allergic andautoimmune diseases can also be contemplated for promoters of chemokinereceptor function if one contemplates the delivery of sufficientcompound to cause the loss of receptor expression on cells through theinduction of chemokine receptor internalization or delivery of compoundin a manner that results in the misdirection of the migration of cells.

In another aspect, the instant invention may be used to evaluate theputative specific agonists or antagonists of a G protein coupledreceptor. The present invention is directed to the use of the compoundof formula (I) in the preparation and execution of screening assays forcompounds that modulate the activity of chemokine receptors.Furthermore, the compound of this invention is useful in establishing ordetermining the binding site of other compounds to chemokine receptors,e.g., by competitive inhibition or as a reference in an assay to compareits known activity to a compound with an unknown activity. Whendeveloping new assays or protocols, the compound according to thepresent invention could be used to test their effectiveness.Specifically, such compound may be provided in a commercial kit, forexample, for use in pharmaceutical research involving the aforementioneddiseases. The compound of the instant invention is also useful for theevaluation of putative specific modulators of the chemokine receptors.In addition, one could utilize the compound of this invention to examinethe specificity of G protein coupled receptors that are not thought tobe chemokine receptors, either by serving as examples of compounds whichdo not bind or as structural variants of compounds active on thesereceptors which may help define specific sites of interaction.

The compound of formula (I) is used to treat or prevent disordersselected from rheumatoid arthritis, osteoarthritis, septic shock,arthrosclerosis, aneurysm, fever, cardiovascular effects, haemodynamicshock, sepsis syndrome, post ischemic reperfusion injury, malaria,Crohn's disease, inflammatory bowel diseases, mycobacterial infection,meningitis, psoriasis, congestive heart failure, fibrotic diseases,cachexia, graft rejection, autoimmune diseases, skin inflammatorydiseases, multiple sclerosis, radiation damage, hyperoxic alveolarinjury, HIV, HIV dementia, non-insulin dependent diabetes mellitus,asthma, allergic rhinitis, atopic dermatitis, idiopathic pulmonaryfibrosis, bullous pemphigoid, helminthic parasitic infections, allergiccolitis, eczema, conjunctivitis, transplantation, familial eosinophilia,eosinophilic cellulitis, eosinophilic pneumonias, eosinophilicfasciitis, eosinophilic gastroenteritis, drug induced eosinophilia,cystic fibrosis, Churg-Strauss syndrome, lymphoma, Hodgkin's disease,colonic carcinoma, Felty's syndrome, sarcoidosis, uveitis, Alzheimer,glomerulonephritis, and systemic lupus erythematosus.

In another aspect, the compound of formula (I) is used to treat orprevent inflammatory disorders selected from rheumatoid arthritis,osteoarthritis, arthrosclerosis, aneurysm, fever, cardiovasculareffects, Crohn's disease, inflammatory bowel diseases, psoriasis,congestive heart failure, multiple sclerosis, autoimmune diseases, skininflammatory diseases.

In another aspect, the compound is used to treat or prevent inflammatorydisorders selected from rheumatoid arthritis, osteoarthritis,arthrosclerosis, Crohn's disease, inflammatory bowel diseases, andmultiple sclerosis.

Combined therapy to prevent and treat inflammatory, infectious andimmunoregulatory disorders and diseases, including asthma and allergicdiseases, as well as autoimmune pathologies such as rheumatoid arthritisand arthrosclerosis, and those pathologies noted above is illustrated bythe combination of the compound of this invention and other compoundswhich are known for such utilities. For example, in the treatment orprevention of inflammation, the present compound may be used inconjunction with an anti-inflammatory or analgesic agent such as anopiate agonist, a lipoxygenase inhibitor, a cyclooxygenase-2 inhibitor,an interleukin inhibitor, such as an interleukin-1 inhibitor, a tumornecrosis factor inhibitor, an NMDA antagonist, an inhibitor or nitricoxide or an inhibitor of the synthesis of nitric oxide, a non-steroidalanti-inflammatory agent, a phosphodiesterase inhibitor, or acytokine-suppressing anti-inflammatory agent, for example with acompound such as acetaminophen, aspirin, codeine, fentanyl, ibuprofen,indomethacin, ketorolac, morphine, naproxen, phenacetin, piroxicam, asteroidal analgesic, sufentanyl, sunlindac, interferon alpha and thelike. Similarly, the instant compound may be administered with a painreliever; a potentiator such as caffeine, an H2-antagonist, simethicone,aluminum or magnesium hydroxide; a decongestant such as phenylephrine,phenylpropanolamine, pseudophedrine, oxymetazoline, ephinephrine,naphazoline, xylometazoline, propylhexedrine, or levodesoxy-ephedrine;and antitussive such as codeine, hydrocodone, caramiphen,carbetapentane, or dextramethorphan; a diuretic; and a sedating ornon-sedating antihistamine. Likewise, the compound of formula (I) may beused in combination with other drugs that are used in thetreatment/prevention/suppression or amelioration of the diseases orconditions for which the compound of the present invention is useful.Such other drugs may be administered, by a route and in an amountcommonly used therefore, contemporaneously or sequentially with acompound of the present invention. When the compound of formula (I) isused contemporaneously with one or more other drugs, a pharmaceuticalcomposition containing such other drugs in addition to the compound offormula (I) may be used. Accordingly, the pharmaceutical compositions ofthe present invention include those that also contain one or more otheractive ingredients, in addition to the compound of formula (I).

Examples of other active ingredients that may be combined with thecompound of the present invention, either administered separately or inthe same pharmaceutical compositions, include, but are not limited to:(a) integrin antagonists such as those for selectins, ICAMs and VLA-4;(b) steroids such as beclomethasone, methylprednisolone, betamethasone,prednisone, dexamethasone, and hydrocortisone; (c) immunosuppressantssuch as cyclosporin, tacrolimus, rapamycin and other FK-506 typeimmunosuppressants; (d) antihistamines (H1-histamine antagonists) suchas bromopheniramine, chlorpheniramine, dexchlorpheniramine,triprolidine, clemastine, diphenhydramine, diphenylpyraline,tripelennamine, hydroxyzine, methdilazine, promethazine, trimeprazine,azatadine, cyproheptadine, antazoline, pheniramine pyrilamine,astemizole, terfenadine, loratadine, cetirizine, fexofenadine,descarboethoxyloratadine, and the like; (e) non-steroidalanti-asthmatics such as b2-agonists (terbutaline, metaproterenol,fenoterol, isoetharine, albuteral, bitolterol, and pirbuterol),theophylline, cromolyn sodium, atropine, ipratropium bromide,leukotriene antagonists (zafirlukast, montelukast, pranlukast,iralukast, pobilukast, SKB-102,203), leukotriene biosynthesis inhibitors(zileuton, BAY-1005); (f) non-steroidal anti-inflammatory agents(NSAIDs) such as propionic acid derivatives (alminoprofen, benxaprofen,bucloxic acid, carprofen, fenbufen, fenoprofen, fluprofen, flurbiprofen,ibuprofen, indoprofen, ketoprofen, miroprofen, naproxen, oxaprozin,pirprofen, pranoprofen, suprofen, tiaprofenic acid, and tioxaprofen),acetic acid derivatives (indomethacin, acemetacin, alclofenac, clidanac,diclofenac, fenclofenac, fenclozic acid, fentiazac, furofenac, ibufenac,isoxepac, oxpinac, sulindac, tiopinac, tolmetin, zidometacin, andzomepirac), fenamic acid derivatives (flufenamic acid, meclofenamicacid, mefenamic acid, niflumic acid and tolfenamic acid),biphenylcarboxylic acid derivatives (diflunisal and flufenisal), oxicams(isoxicam, piroxicam, sudoxicam and tenoxican), salicylates (acetylsalicylic acid, sulfasalazine) and the pyrazolones (apazone,bezpiperylon, feprazone, mofebutazone, oxyphenbutazone, phenylbutazone);(g) cyclooxygenase-2 (COX-2) inhibitors; (h) inhibitors ofphosphodiesterase type IV (PDE-IV); (i) other antagonists of thechemokine receptors; (j) cholesterol lowering agents such as HMG-COAreductase inhibitors (lovastatin, simvastatin and pravastatin,fluvastatin, atorvastatin, and other statins), sequestrants(cholestyramine and colestipol), nicotonic acid, fenofibric acidderivatives (gemfibrozil, clofibrat, fenofibrate and benzafibrate), andprobucol; (k) anti-diabetic agents such as insulin, sulfonylureas,biguanides (metformin), a-glucosidase inhibitors (acarbose) andglitazones (troglitazone and pioglitazone); (l) preparations ofinterferons (interferon alpha-2a, interferon-2B, interferon alpha-N3,interferon beta-1a, interferon beta-1b, interferon gamma-1b); (m)antiviral compounds such as efavirenz, nevirapine, indinavir,ganciclovir, lamivudine, famciclovir, and zalcitabine; (n) othercompound such as 5-aminosalicylic acid and prodrugs thereof,anti-metabolites such as azathioprine and 6-mercaptopurine, andcytotoxic cancer chemotherapeutic agents. The weight ratio of thecompound of formula (I) to the second active ingredient may be variedand will depend upon the effective doses of each ingredient.

Generally, an effective dose of each will be used. Thus, for example,when the compound of formula (I) is combined with an NSAID the weightratio of the compound of the present invention to the NSAID willgenerally range from about 1000:1 to about 1:1000, or alternatively fromabout 200:1 to about 1:200. Combinations of the compound of formula (I)and other active ingredients will generally also be within theaforementioned range, but in each case, an effective dose of each activeingredient should be used.

The compound is administered to a mammal in a therapeutically effectiveamount. By “therapeutically effective amount” it is meant an amount ofthe compound of Formula I that, when administered alone or incombination with an additional therapeutic agent to a mammal, iseffective to prevent or ameliorate the thromboembolic disease conditionor the progression of the disease.

Dosage and Formulation

The compound of this invention can be administered in such oral dosageforms as tablets, capsules (each of which includes sustained release ortimed release formulations), pills, powders, granules, elixirs,tinctures, suspensions, syrups, and emulsions. It may also beadministered in intravenous (bolus or infusion), intraperitoneal,subcutaneous, or intramuscular form, all using dosage forms well knownto those of ordinary skill in the pharmaceutical arts. It can beadministered alone, but generally will be administered with apharmaceutical carrier selected on the basis of the chosen route ofadministration and standard pharmaceutical practice.

The dosage regimen for the compound of the present invention will, ofcourse, vary depending upon known factors, such as the pharmacodynamiccharacteristics of the particular agent and its mode and route ofadministration; the species, age, sex, health, medical condition, andweight of the recipient; the nature and extent of the symptoms; the kindof concurrent treatment; the frequency of treatment; the route ofadministration, the renal and hepatic function of the patient, and theeffect desired. A physician or veterinarian can determine and prescribethe effective amount of the drug required to prevent, counter, or arrestthe progress of the thromboembolic disorder.

By way of general guidance, the daily oral dosage of each activeingredient, when used for the indicated effects, will range betweenabout 0.001 to 1000 mg/kg of body weight, or between about 0.01 to 100mg/kg of body weight per day, or alternatively, between about 1.0 to 20mg/kg/day. Intravenously, the doses will range from about 1 to about 10mg/kg/minute during a constant rate infusion. The compound of thisinvention may be administered in a single daily dose, or the total dailydosage may be administered in divided doses of two, three, or four timesdaily.

The compound of this invention can be administered in intranasal formvia topical use of suitable intranasal vehicles, or via transdermalroutes, using transdermal skin patches. When administered in the form ofa transdermal delivery system, the dosage administration will, ofcourse, be continuous rather than intermittent throughout the dosageregimen.

The compound is typically administered in admixture with suitablepharmaceutical diluents, excipients, or carriers (collectively referredto herein as pharmaceutical carriers) suitably selected with respect tothe intended form of administration, that is, oral tablets, capsules,elixirs, syrups and the like, and consistent with conventionalpharmaceutical practices.

For instance, for oral administration in the form of a tablet orcapsule, the active drug component can be combined with an oral,non-toxic, pharmaceutically acceptable, inert carrier such as lactose,starch, sucrose, glucose, methyl cellulose, magnesium stearate,dicalcium phosphate, calcium sulfate, mannitol, sorbitol and the like;for oral administration in liquid form, the oral drug components can becombined with any oral, non-toxic, pharmaceutically acceptable inertcarrier such as ethanol, glycerol, water, and the like. Moreover, whendesired or necessary, suitable binders, lubricants, disintegratingagents, and coloring agents can also be incorporated into the mixture.Suitable binders include starch, gelatin, natural sugars such as glucoseor beta-lactose, corn sweeteners, natural and synthetic gums such asacacia, tragacanth, or sodium alginate, carboxymethylcellulose,polyethylene glycol, waxes, and the like. Lubricants used in thesedosage forms include sodium oleate, sodium stearate, magnesium stearate,sodium benzoate, sodium acetate, sodium chloride, and the like.Disintegrators include, without limitation, starch, methyl cellulose,agar, bentonite, xanthan gum, and the like.

The compound of the present invention can also be administered in theform of liposome delivery systems, such as small unilamellar vesicles,large unilamellar vesicles, and multilamellar vesicles. Liposomes can beformed from a variety of phospholipids, such as cholesterol,stearylamine, or phosphatidylcholines.

The compound of the present invention may also be coupled with solublepolymers as targetable drug carriers. Such polymers can includepolyvinylpyrrolidone, pyran copolymer,polyhydroxypropylmethacrylamide-phenol,polyhydroxyethylaspartamidephenol, or polyethyleneoxide-polylysinesubstituted with palmitoyl residues. Furthermore, the compounds of thepresent invention may be coupled to a class of biodegradable polymersuseful in achieving controlled release of a drug, for example,polylactic acid, polyglycolic acid, copolymers of polylactic andpolyglycolic acid, polyepsilon caprolactone, polyhydroxy butyric acid,polyorthoesters, polyacetals, polydihydropyrans, polycyanoacylates, andcrosslinked or amphipathic block copolymers of hydrogels.

Dosage forms (pharmaceutical compositions) suitable for administrationmay contain from about 1 milligram to about 100 milligrams of activeingredient per dosage unit. In these pharmaceutical compositions theactive ingredient will ordinarily be present in an amount of about0.5-95% by weight based on the total weight of the composition.

Gelatin capsules may contain the active ingredient and powderedcarriers, such as lactose, starch, cellulose derivatives, magnesiumstearate, stearic acid, and the like. Similar diluents can be used tomake compressed tablets. Both tablets and capsules can be manufacturedas sustained release products to provide for continuous release ofmedication over a period of hours. Compressed tablets can be sugarcoated or film coated to mask any unpleasant taste and protect thetablet from the atmosphere, or enteric coated for selectivedisintegration in the gastrointestinal tract.

Liquid dosage forms for oral administration can contain coloring andflavoring to increase patient acceptance.

In general, water, a suitable oil, saline, aqueous dextrose (glucose),and related sugar solutions and glycols such as propylene glycol orpolyethylene glycols are suitable carriers for parenteral solutions.Solutions for parenteral administration may contain a water soluble saltof the active ingredient, suitable stabilizing agents, and if necessary,buffer substances. Antioxidizing agents such as sodium bisulfate, sodiumsulfite, or ascorbic acid, either alone or combined, are suitablestabilizing agents. Also used are citric acid and its salts and sodiumEDTA. In addition, parenteral solutions can contain preservatives, suchas benzalkonium chloride, methyl- or propyl-paraben, and chlorobutanol.

Suitable pharmaceutical carriers are described in Remington'sPharmaceutical Sciences, Mack Publishing Company, a standard referencetext in this field.

Representative useful pharmaceutical dosage-forms for administration ofthe compounds of this invention can be illustrated as follows:

Capsules

A large number of unit capsules can be prepared by filling standardtwo-piece hard gelatin capsules each with 100 milligrams of powderedactive ingredient, 150 milligrams of lactose, 50 milligrams ofcellulose, and 6 milligrams magnesium stearate.

Soft Gelatin Capsules

A mixture of active ingredient in a digestible oil such as soybean oil,cottonseed oil or olive oil may be prepared and injected by means of apositive displacement pump into gelatin to form soft gelatin capsulescontaining 100 milligrams of the active ingredient. The capsules shouldbe washed and dried.

Tablets

Tablets may be prepared by conventional procedures so that the dosageunit is 100 milligrams of active ingredient, 0.2 milligrams of colloidalsilicon dioxide, 5 milligrams of magnesium stearate, 275 milligrams ofmicrocrystalline cellulose, 11 milligrams of starch and 98.8 milligramsof lactose. Appropriate coatings may be applied to increase palatabilityor delay absorption.

Injectable

A parenteral composition suitable for administration by injection may beprepared by stirring 1.5% by weight of active ingredient in 10% byvolume propylene glycol and water. The solution should be made isotonicwith sodium chloride and sterilized.

Suspension

An aqueous suspension can be prepared for oral administration so thateach 5 mL contain 100 mg of finely divided active ingredient, 200 mg ofsodium carboxymethyl cellulose, 5 mg of sodium benzoate, 1.0 g ofsorbitol solution, U.S.P., and 0.025 mL of vanillin.

Where the compound of this invention is combined with otheranticoagulant agents, for example, a daily dosage may be about 0.1 to100 milligrams of the compound of Formula I and about 1 to 7.5milligrams of the second anticoagulant, per kilogram of patient bodyweight. For a tablet dosage form, the compound of this inventiongenerally may be present in an amount of about 5 to 10 milligrams perdosage unit, and the second anti-coagulant in an amount of about 1 to 5milligrams per dosage unit.

Where two or more of the foregoing second therapeutic agents areadministered with the compound of Formula I, generally the amount ofeach component in a typical daily dosage and typical dosage form may bereduced relative to the usual dosage of the agent when administeredalone, in view of the additive or synergistic effect of the therapeuticagents when administered in combination. Particularly when provided as asingle dosage unit, the potential exists for a chemical interactionbetween the combined active ingredients. For this reason, when thecompound of Formula I and a second therapeutic agent are combined in asingle dosage unit they are formulated such that although the activeingredients are combined in a single dosage unit, the physical contactbetween the active ingredients is minimized (that is, reduced). Forexample, one active ingredient may be enteric coated. By enteric coatingone of the active ingredients, it is possible not only to minimize thecontact between the combined active ingredients, but also, it ispossible to control the release of one of these components in thegastrointestinal tract such that one of these components is not releasedin the stomach but rather is released in the intestines. One of theactive ingredients may also be coated with a material which effects asustained-release throughout the gastrointestinal tract and also servesto minimize physical contact between the combined active ingredients.Furthermore, the sustained-released component can be additionallyenteric coated such that the release of this component occurs only inthe intestine. Still another approach would involve the formulation of acombination product in which the one component is coated with asustained and/or enteric release polymer, and the other component isalso coated with a polymer such as a low-viscosity grade ofhydroxypropyl methylcellulose (HPMC) or other appropriate materials asknown in the art, in order to further separate the active components.The polymer coating serves to form an additional barrier to interactionwith the other component.

These as well as other ways of minimizing contact between the componentsof combination products of the present invention, whether administeredin a single dosage form or administered in separate forms but at thesame time by the same manner, will be readily apparent to those skilledin the art.

While the invention has been described in detail and with reference tospecific embodiments thereof, it will be apparent to one skilled in theart that various changes and modifications can be made therein withoutdeparting from the spirit and scope thereof.

1. A compound of Formula (I):

or a stereoisomer or pharmaceutically acceptable salt form thereof.
 2. Apharmaceutical composition comprised of a pharmaceutically acceptablecarrier and a therapeutically effective amount of the compound ofclaim
 1. 3. A method for modulation of chemokine or chemokine receptoractivity comprising administering to a patient in need thereof atherapeutically effective amount of the compound of claim
 1. 4. Themethod of claim 3, wherein the chemokine or chemokine receptor activityis CCR-1 or CCR-1 receptor activity.
 5. A method for treating a disordercomprising administering to a patient in need thereof a therapeuticallyeffective amount of the compound of claim 1; wherein said disorder isselected from the group consisting of osteoarthritis, aneurysm, fever,cardiovascular effects, Crohn's disease, congestive heart failure,autoimmune diseases, HIV-infection, HIV-associated dementia, psoriasis,idiopathic pulmonary fibrosis, transplant arteriosclerosis, physically-or chemically-induced brain trauma, neuropathic pain, inflammatory boweldisease, alveolitis, ulcerative colitis, systemic lupus erythematosus,nephrotoxic serum nephritis, glomerulonephritis, asthma, multiplesclerosis, arthrosclerosis, rheumatoid arthritis, restenosis, organtransplantation, multiple myeloma, colorectal cancer, hepatocellularcancer and other cancers.
 6. A method for treating inflammatory diseasescomprising administering to a patient in need thereof a therapeuticallyeffective amount of the compound of claim
 1. 7. A method for preparing amedicament for the treatment of osteoarthritis, aneurysm, fever,cardiovascular effects, Crohn's disease, congestive heart failure,autoimmune diseases, HIV-infection, HIV-associated dementia, psoriasis,idiopathic pulmonary fibrosis, transplant arteriosclerosis, physically-or chemically-induced brain trauma, neuropathic pain, inflammatory boweldisease, alveolitis, ulcerative colitis, systemic lupus erythematosus,nephrotoxic serum nephritis, glomerulonephritis, asthma, multiplesclerosis, arthrosclerosis, or rheumatoid arthritis comprisingformulating the compound of claim
 1. 8. A method for treating a patientin need of therapy comprising administering to said patient atherapeutically effective amount of a compound of claim
 1. 9. A methodfor modulation of chemokine or chemokine receptor activity comprisingadministering to a patient in need thereof a therapeutically effectiveamount of a composition of claim
 2. 10. A method for modulation of CCR-1receptor activity comprising administering to a patient in need thereofa therapeutically effective amount of a composition of claim
 2. 11. Amethod for treating a disorder comprising administering to a patient inneed thereof a therapeutically effective amount of a composition ofclaim 2, wherein said disorder is selected from the group consisting ofosteoarthritis, aneurysm, fever, cardiovascular effects, Crohn'sdisease, congestive heart failure, autoimmune diseases, HIV-infection,HIV-associated dementia, psoriasis, idiopathic pulmonary fibrosis,transplant arteriosclerosis, physically- or chemically-induced braintrauma, neuropathic pain, inflammatory bowel disease, alveolitis,ulcerative colitis, systemic lupus erythematosus, nephrotoxic serumnephritis, glomerulonephritis, asthma, multiple sclerosis,arthrosclerosis, rheumatoid arthritis, restenosis, organtransplantation, multiple myeloma, colorectal cancer, hepatocellularcancer and other cancers.
 12. A method for treating inflammatorydiseases comprising administering to a patient in need thereof atherapeutically effective amount of a composition of claim
 2. 13. Amethod for preparing a medicament for the treatment of osteoarthritis,aneurysm, fever, cardiovascular effects, Crohn's disease, congestiveheart failure, autoimmune diseases, HIV-infection, HIV-associateddementia, psoriasis, idiopathic pulmonary fibrosis, transplantarteriosclerosis, physically- or chemically-induced brain trauma,neuropathic pain, inflammatory bowel disease, alveolitis, ulcerativecolitis, systemic lupus erythematosus, nephrotoxic serum nephritis,glomerulonephritis, asthma, multiple sclerosis, arthrosclerosis, orrheumatoid arthritis comprising formulating a composition of claim 2into a useful pharmaceutical dosage-form.
 14. A method for treating apatient in need of therapy comprising administering to said patient atherapeutically effective amount of a composition of claim 2.